Animal cells have both a proliferating phase and a quiescent phase. Cells can shift from the proliferating phase to the quiescent phase during a brief window in the cell cycle. Depending on their position in the cell cycle, cells deprived of mitogens such as those present in serum can undergo immediate cell cycle arrest, or they can complete the current mitotic cycle and arrest in the next cell cycle. The transition from mitogen-dependence to mitogen-independence occurs in mid- to late-G1 phase of the cell cycle. Anti-mitogenic signals can cause the cell cycle to arrest at a kinetically common point. In particular, in early G1, cells can exit the cell cycle. Cell cycle commitment (autonomy from mitogenic signals) occurs in mid-G1.
The transition of cells through G1 and entry into S phase requires the action of cyclin-dependent kinases (Cdks). Growth inhibitory signals have been shown to prevent activation of these Cdks during G1 (Serrano et al., Nature 366:704-07 (1993); Hannon and Beach, Nature 371:257-61 (1994); Xiong et al., Nature 366:701-04 (1993); Polyak et al., Cell 78:59-66 (1994); Lee et al., Genes & Development 9:639-49 (1995); Koff et al., Science 260:536-39 (1993)). The catalytic activity of Cdks is known to be regulated by two general mechanisms: protein phosphorylation and association with regulatory subunits (Gould et al., EMBO J. 10:3297-309 (1991); Solomon et al., EMBO J. 12:3133-42 (1993); Solomon et al., Mol. Biol. Cell 3:13-27 (1992); Jeffrey et al., Nature 376:313-20 (1995); Morgan, Nature 374:131-34 (1995)). Among the regulatory subunits, the association of Cdks with inhibitory CKI subunits (Cyclin-dependent Kinase Inhibitors) has been most closely correlated with the effect of mitogen depletion on cell proliferation and Cdk activity.
The CKI directly implicated in mitogen-dependent Cdk regulation is p27Kip1 (Polyak et al., Cell 78:59-66 (1994); Toyoshima and Hunter, Cell 78:67-77 (1994)). Wildtype p27Kip1 protein accumulates to high levels in quiescent cells, and is rapidly destroyed after quiescent cells are re-stimulated with specific mitogens (Nourse et al., Nature 372:570-73 (1994); Kato et al., Cell 79:487-96 (1994)). The destruction of p27Kip1 is controlled by phosphorylation of p27Kip1 at threonine 187 (T187). T187 is phosphorylated by Cdk2 to create a binding site for a Skp2-containing ubiquitin-protein ligase known as the Skp1-cullin-F-box protein ligase (SCF) (Feldman et al., Cell 91:221-30 (1997); Bai et al., Cell 86:263-74 (1996); Skowyra et al., Cell 91:209-19 (1997)). Ubiquitination of p27Kip1 by the SCF then results in p27Kip1 degradation by the proteosome (Sutterluty et al., Nature Cell Biol. 1:207-14 (1999); Rolfe et al., J. Mol. Med. 75:5-17 (1997); Carrano et al., Nature Cell Biol. 1:193-99 (1999); Tsvetkov et al., Curr. Biol. 9:661-64 (1999)).
The destruction of p27Kip1 was thought to be required for entry into S phase. Moreover, constitutive expression of p27Kip1 in cultured cells causes the cell cycle to arrest in G1 (Polyak, supra; Toyoshima and Hunter, supra). Thus, based on these observations, it was expected that cells harboring a null allele of p27Kip1 would arrest G1. It was surprising, therefore, that animals harboring a null allele of the p27Kip1 gene survived. Indeed, such animals were larger than normal (increased animal size) and without apparent gross morphologic abnormalities. (Fero et al., Cell 85:733-44 (1996); U.S. Pat. No. 5,958,769; the disclosures of which are incorporated by reference herein.) The advantages of producing larger animals are readily apparent, and include increase meat, milk and/or egg production.
Decreased levels of p27Kip1 in animals, however, cause certain minor defects, such as an ovulatory defect, and resulting female sterility, increased pituitary tumorigenesis and disrupted retinal architecture. (Fero et al., supra.) These defects can interfere with some uses of such animals. Thus, there is a need for alternative mutant alleles of p27Kip1, and of methods of using such mutant alleles, that promote increased animal size or growth rate without these side effects.